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82505318.Pdf View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Am. J. Hum. Genet. 70:1568–1574, 2002 Report The Gene Encoding Nicastrin, a Major g-Secretase Component, Modifies Risk for Familial Early-Onset Alzheimer Disease in a Dutch Population-Based Sample Bart Dermaut,1 Jessie Theuns,1 Kristel Sleegers,2 Hiroshi Hasegawa,3 Marleen Van den Broeck,1 Krist’l Vennekens,1 Ellen Corsmit,1 Peter St. George-Hyslop,3 Marc Cruts,1 Cornelia M. van Duijn,1,2 and Christine Van Broeckhoven1 1Department of Molecular Genetics, Flanders Interuniversity Institute of Biotechnology, University of Antwerp (Universitaire Instelling Antwerpen), Antwerp; 2Department of Epidemiology and Biostatistics, Erasmus Medical Center, Rotterdam; and 3Centre for Research in Neurodegenerative Diseases and Department of Medicine, University of Toronto, Department of Medicine (Neurology), Toronto Western Hospital, University of Toronto, Toronto Nicastrin regulates g-secretase cleavage of the amyloid precursor protein by forming complexes with presenilins, in which most mutations causing familial early-onset Alzheimer disease (EOAD) have been found. The gene encoding nicastrin (NCSTN) maps to 1q23, a region that has been linked and associated with late-onset Alzheimer disease (LOAD) in various genome screens. In 78 familial EOAD cases, we found 14 NCSTN single-nucleotide polymor- phisms (SNPs): 10 intronic SNPs, 3 silent mutations, and 1 missense mutation (N417Y). N417Y is unlikely to be pathogenic, since it did not alter amyloid b secretion in an in vitro assay and its frequency was similar in case and control subjects. However, SNP haplotype estimation in two population-based series of Dutch patients with EOAD (n p 116 ) and LOAD (n p 240 ) indicated that the frequency of one SNP haplotype (HapB) was higher in the group with familial EOAD (7%), compared with the LOAD group (3%) and control group (3%). In patients with familial EOAD without the APOE e4 allele, the HapB frequency further increased, to 14%, resulting in a fourfold increased risk (odds ratio p 4.1; 95% confidence interval 1.2–13.3;P p .01 ). These results are compatible with an important role of g-secretase dysfunction in the etiology of familial EOAD. The amyloid b peptide (Ab) is the main component of causative EOAD mutations directly influence g-secretase the neuritic plaque, the histological hallmark lesion in activity, at the level of either the substrate (APP muta- the brains of patients with Alzheimer disease (AD [MIM tions at the g cleavage site) or the enzyme (all PSEN 104300]). Ab is generated by b- and subsequent g-se- mutations), g-secretase dysfunction is crucial in familial cretase cleavage of the amyloid precursor protein (APP EOAD pathogenesis. Recent findings by us (van Duijn [MIM 104760]). Mutations in APP and the presenilins et al. 1999; Theuns et al. 2000) and others (Lambert et (PSEN1 [MIM 104311] and PSEN2 [MIM 600759]) al. 2001), of genetic association between the promot- cause the rare familial early-onset type of AD (EOAD) er region of PSEN1 and EOAD, further emphasize the (AD Mutation Database). Since the large majority of importance of altered g-secretase activity in EOAD. Recently, a novel type 1 transmembrane glycoprotein, Received January 9, 2002; accepted for publication March 11, 2002; designated “nicastrin” (NCSTN [MIM 605254]), was electronically published April 24, 2002. identified as a component of the presenilin-containing Address for correspondence and reprints: Dr. Christine Van Broeck- g-secretase complex that is involved in the cleaving of hoven, Department of Molecular Genetics (VIB8), University of An- APP and Notch in their transmembrane domains (Yu et twerp (UIA), Universiteitsplein 1, B-2610, Antwerpen, Belgium. E- al. 2000; Chen et al. 2001). Like all pathogenic PSEN mail: [email protected] ᭧ 2002 by The American Society of Human Genetics. All rights reserved. mutations, missense mutations in a conserved hydro- 0002-9297/2002/7006-0019$15.00 philic domain (DYIGSrAAIGS) in NCSTN strongly in- 1568 Reports 1569 Figure 1 Genomic structure of NCSTN and major SNP haplotypes, with their estimated frequencies in 78 patients with familial EOAD. SNPs used for case-control association analysis are in boldface. creased Ab production, whereas deletions in this domain in 78 patients was classified as familial (i.e., at least one inhibited Ab production (Yu et al. 2000). NCSTN maps first-degree relative with dementia), 10 cases of which to 1q23, a region that has shown evidence for linkage fulfilled our criteria of autosomal dominant inheritance to (Kehoe et al. 1999) and association with LOAD (Hil- (Cruts et al. 1998). In this sample, seven patients were tunen et al. 2001). identified who carried a causative PSEN mutation (five We evaluated the contribution of genetic variations in in PSEN1 and two in PSEN2) (Cruts et al. 1998; authors’ NCSTN in two large series of patients with EOAD (onset unpublished data). Patients with EOAD were compared before or at age 65 years) and LOAD (onset after age with a control group of 114 individuals in the same age years; 46% 3.3 ע years). Patients with EOAD were derived from a range (mean age at examination60.6 65 Dutch population-based study of EOAD in the four male). Patients with LOAD were drawn from the Rot- northern provinces of the Netherlands and metropolitan terdam study, a population-based prospective study on Rotterdam (van Duijn et al. 1994a). The patients were residents of age у55 years from a Rotterdam suburb in sampled during two study periods. The original sample the Netherlands (Hofman et al. 1991). Dementia was was collected between 1980 and 1987 and has been diagnosed by a three-step examination, as detailed else- described elsewhere (van Duijn et al. 1994b). The initial where (Ott et al. 1995). The diagnosis of probable AD study was extended between 1997 and 2000, in a ge- was based on NINCDS-ADRDA criteria (McKhann et netically isolated part of the previously described area, al. 1984): 339 patients with AD were ascertained at the with the same sampling criteria. The current sample start of the study, and 116 patients with incident AD comprises 96 patients from the original group, with an were diagnosed at the first follow-up period. DNA was additional 20 patients from the extended study. Mean available for 240 patients with probable LOAD cases -years; 20% male). Pa 6.9 ע years, and 23% of patients (mean age at onset84.1 5.5 ע age at onset was56.4 in the sample were male. The diagnosis was indepen- tients with LOAD were compared to 239 control indi- ;years 3.7 ע dently confirmed by a member of the research team and viduals (mean age at examination72.1 by a neurologist using a standardized protocol consistent 15% male). All control individuals were taken from the with the National Institute of Neurological and Com- Rotterdam study and were screened for cognitive dys- municative Disorders and Stroke-Alzheimer’s Disease function. Potential control subjects with dementia were and Related Disorders Association (NINCDS-ADRDA) excluded. criteria for AD (McKhann et al. 1984). The disease in We determined the genomic organization of NCSTN 38 patients was classified as sporadic, and the disease (gDNA: 15.9 kb; cDNA: 2.9 kb) by alignment of the 1570 Am. J. Hum. Genet. 70:1568–1574, 2002 complete cDNA sequence (NCSTN cDNA [GenBank Table 1 accession number AF240468]) against the human work- NCSTN Sequence Variations and Their Allele Frequencies in 78 ing draft sequence (Human Genome Project Work- Familial EOAD Cases ing Draft Web site). NCSTN consists of 17 exons (fig. No. (Frequency) 1), all of which contain coding sequence. Using PCR Location Polymorphism of Mutant Allelea primers flanking each exon, we screened NCSTN exons Exon 3 c.237GrA (Glu79) 2 (.01) 1–16 and the coding part of exon 17 by denaturing high- Intron 4 IVS4Ϫ69CrG 1 (.01) performance liquid chromatography (DHPLC) in 78 pa- Intron 5 IVS5Ϫ74CrT 13 (.08) tients with familial AD, including 10 patients with au- Exon 6 c.636ArG (Leu212) 13 (.08) tosomal dominant EOAD. Direct sequencing of PCR Intron 6 IVS6ϩ18CrG 8 (.05) r fragments with aberrant DHPLC patterns revealed 14 Exon 7 c.747C T (Asp249) 13 (.08) Intron 7 IVS7ϩ61GrA 2 (.01) single nucleotide polymorphisms (SNPs), of which 10 Intron 10 IVS10ϩ32CrG 1 (.01) were located in introns and 4 in coding regions (table Intron 10 IVS10Ϫ5CrG 13 (.08) 1). Direct sequencing of NCSTN in the probands of the Exon 11 c.1249ArT (Asn417Tyr) 1 (.01) 10 autosomal dominant families revealed no causative Intron 13 IVS13Ϫ129TrC 16 (.10) r Intron 15 IVS15ϩ31insG 1 (.01) mutations or additional variations. Only c.1249A T Ϫ r r Intron 16 IVS16 119G C 16 (.10) predicts an amino acid substitution (N Y at codon 417) Intron 16 IVS16Ϫ74CrT 3 (.02) and was found in a patient with familial EOAD (age at a p onset 57 years). No other family members were available Frequencies are given as proportion ofN 156 total alleles. for segregation analysis. NCSTN N417Y is a conser- vative amino acid substitution affecting a residue that is not evolutionarily conserved in mouse (A), Drosophila as “HapA,” “HapB,” “HapC,” and “HapD”) (fig. 1). (E), or Caenorhabditis elegans (G) (Yu et al. 2000) but Through use of a pyrosequencing assay, genotypes for is located within a putative glycosylation site (N-Q-S). each of these four SNPs were determined in patients To test the potential contribution of N417Y to AD, we with EOAD, patients with LOAD, and with their age- used a pyrosequencing assay (Alderborn et al. 2000; matched control groups (table 2). Genotype frequencies Theuns et al. 2001) to screen all patients with EOAD, for each SNP were in Hardy-Weinberg equilibrium as well as 235 patients with probable LOAD and a total (HWE) in all case and control groups (P 1 .23 ).
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